Cathelicidin Enhances the LPS-Inducing Synthesis of Toll-like Receptors 4 in the Colonic Epithelium

MARIN MS; HOLANI R; SHAH C; HAJI Q; ODEÓN AC; COBO E

FASEB JOURNAL

FEDERATION AMER SOC EXP BIOL

Año: 2016 vol. 30

0892-6638

Cathelicidins are innate antimicrobial peptides with broad immunomodulatory functions however; their role in modulating intestinal defenses remains elusive. This work investigated whether cathelicidins modulate the synthesis of Toll-like receptors (TLRs) in the intestinal epithelium. Human colonic adenocarcinoma epithelial cells (HT29) were stimulated with inducers of endogenous cathelicidins (sodium butyrate and vitamin D) or synthetic human cathelicidin (LL-37) in association with agonists/antagonists of TLR4 and 9. Total RNA from cells was isolated prior to cDNA synthesis. Gene expression of human TLR4, TLR9 (RT2 qPCR Primer Assay, Qiagen) and a housekeeping gene (GAPDH) were quantified by real time RT-qPCR and data analyzed using the 2−ΔΔCT method. Protein synthesis of TLRs was assessed by western blotting in which the cell lysates were separated on 4?15% TGX stain-free gels (BioRad), transferred to PVDF membrane, blocked and incubated with anti-TLR4 or anti-TLR9 antibody (Abcam). Densitometry was analyzed (ImageLab, BioRad) and normalized with reference to the total lane protein. Antimicrobial activity in HT29 cell lysates challenged with Escherichia coli and stimulated with LPS and LL-37 was analyzed by bacterial counting. For statistical analysis between treatment groups the paired two tailed Student?s t-test was used. We observed that butyrate induced TLR4 gene expression alone, as well as in synergy with TLR4 agonist LPS (Fig 1A). In addition, simultaneous exogenous LL-37 and LPS induced higher expression of TLR4 than LPS alone (Fig 2A). The synergic effect of cathelicidin and LPS in the TLR4 mRNA expression was confirmed in colonocytes pre-treated with TLR4 antagonist LPS-RS (Fig 3). Likewise, we found that LPS and butyrate, or LL-37 directly increased the TLR4 protein synthesis (Fig 4A). A stimulating effect of butyrate or cathelicidins was not seen on TLR9. Herein, the TLR9 agonist CpG ODN only induced TLR9 synthesis and the addition of butyrate or LL-37 countered the CpG ODN induced TLR9 overexpression at gene (Fig 1B and 2B) and protein levels (Fig 4B). Co-stimulation with LPS and LL-37 showed increased antimicrobial activity (~50% inhibition) compared with LPS only (Fig 5A). Likewise, TLR9 agonist alone or with LL-37 reduced the number of surviving E. coli (Fig 5A). TLR agonists and LL-37 showed a similar antimicrobial activity in E. coli challenged HT29 cell supernatants (Fig 5B). We conclude that exogenous cathelicidins (and likely butyrate induced endogenous cathelicidins) in association with LPS induces TLR4 mRNA and protein in colonocytes. Overexpression of intestinal epithelial TLR4 (minimally expressed and hypo-responsive to LPS under physiologic conditions), is necessary during intestinal inflammation for epithelial restoration and integrity. It could be accompanied by suppression of agonist mediated TLR9 stimulation. Thus, cathelicidin is key in intestinal homeostasis as it modulates the priming provided by TLR ligands and the synthesis of TLR4 and 9 in the intestinal epithelium.